Inhibition of cellular HIV-1 protease activity by lysyl-tRNA synthetase.

During early assembly of human immunodeficiency virus type 1 (HIV-1), an assembly complex is formed, the components of which include genomic RNA, Gag, GagPol, tRNA(Lys), and lysyl tRNA synthetase (LysRS). Directly increasing or decreasing cellular expression of LysRS results in corresponding changes in viral infectivity and in the viral concentrations of LysRS, tRNA(Lys), and, surprisingly, reverse transcriptase (RT). Since altering the cellular expression of LysRS does not lead to a change in the incorporation of the RT precursor protein, GagPol, in protease-negative HIV-1, we propose that the altered viral content of RT resulting from alterations in cellular LysRS concentration results from the ability of LysRS to inhibit premature activation of Gag-Pol viral protease within the complex. Supporting this hypothesis, we find that increases and decreases in cellular LysRS expression are accompanied by 5-8-fold increases and 5-fold decreases, respectively, in the cytoplasmic proteolysis of Gag and GagPol to mature viral proteins. Using a novel bioluminescence resonance energy transfer assay to directly measure HIV-1 protease activity in vivo also indicates that the overexpression of LysRS in the cell reduces viral protease activity.

During viral assembly, the Gag and GagPol precursors are processed into the final mature viral proteins by the viral protease located within GagPol (1). Proteolysis is believed to occur during or immediately after viral budding (2), and mechanisms that prevent premature cytoplasmic processing of the viral precursors and their loss from the virion are clearly desirable. Since protease dimerization is required for the activity of the enzyme (1), the concentration of GagPol at the plasma membrane could be an important factor favoring dimerization. Another mechanism regulating protease activity could involve a GagPol conformation, rendering it resistant to protease dimerization during its transit to the plasma membrane. Such a GagPol transport complex will include Gag and RNA since interaction with Gag is required for GagPol incorporation into budding viruses (3,4), and this interaction requires the RNA-facilitated multimerization of Gag (5).
This RNA/Gag/GagPol complex will also include tRNA Lys and lysyl tRNA synthetase (LysRS), the enzyme that aminoacylates tRNA Lys . During HIV-1 1 assembly, both LysRS (6) and the tRNA Lys isoacceptors, tRNA 1 Lys , tRNA 2 Lys , and tRNA 3 Lys (the primer tRNA for reverse-transcriptase (7)), are selectively packaged into HIV-1 (8). This incorporation requires the presence of both Gag, which binds specifically with LysRS (9), and GagPol, which interacts with both Gag (3,4) and tRNA Lys (10). LysRS incorporation into Gag viral-like particles occurs independently of tRNA Lys incorporation (6), which requires the additional presence of GagPol, suggesting that LysRS itself may act as a signal for targeting tRNA Lys for selective packaging into HIV-1. A complex containing Gag, GagPol, and LysRS has been coimmunoprecipitated with anti-integrase from lysates of 293FT cells infected with protease-negative HIV-1 (9).
GagPol present in an early form of this assembly complex may be more resistant to dimerization and protease activation than at later stages when budding is initiated. In support of this hypothesis, we present data herein that indicate that altering the content of one of the packaging complex components, LysRS, produces corresponding changes in viral protease activity.

EXPERIMENTAL PROCEDURES
Plasmids-BH10 is a simian virus 40-based vector that contains full-length wild-type HIV-1 proviral DNA. BH10.P-is similar to BH10 but contains an inactive viral protease (D25G). hGagPol codes for Gag and protease-positive GagPol. hGagPol⌬FS⌬PR was constructed by deleting 5 thymidines in the frameshift site and codes for GagPol but not Gag. This protein contains an inactive protease due to an R42G mutation in the active site. Both proteins are made from mRNAs that have had their codons optimized for mammalian cell codon usage. The "humanized" proteins have identical amino acid sequences to their viral counterparts. pcDNALysRS contains cDNA encoding full-length (1-597 amino acids) human LysRS cloned into pcDNA3.1 (Invitrogen) and was constructed through PCR amplification of the cDNA as described previously (11). It is C-terminally tagged with V5. BH10Lys3 and BH10Lys2 contain both wild-type HIV-1 proviral DNA and a human tRNA 3 Lys or tRNA 2 Lys gene, respectively. These vectors were constructed as described previously (12). For bioluminescence resonance energy transfer (BRET) analysis, a DNA construct was prepared coding for a fusion protein between humanized sea pansy Renilla reniformis Luciferase (hRLuc2) and a humanized green fluorescent protein (hGFP2). The HIV-1 p2/p7 protease cleavage site (4 amino acids on each side of * This work was supported in part by grants from the National Institutes of Health and the Canadian Institutes for Health Research (CIHR) and the Canadian Foundation for Innovation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the scissile bond: ATIM/MQRG) was fused between and in-frame to create the hGFP2-p2/p7-hRLuc construct, as described (13).
Cell Transfections, siRNA, and Virus Purification-The culture of HEK-293T cells, their transfection with these plasmids using Lipofectamine 2000 (Invitrogen), and the isolation of virions 48 h after transfection from the cell supernatant were done as described previously (14). siRNA experiments were performed as described previously (15). 293T cells were first transfected with small interfering RNA specific for either LysRS (siRNA LysRS ) or a control small interfering RNA siRNA Luc ) specific for luciferase, and 24 h later, transfected with BH10. Viruses were harvested 48 h after transfection with BH10. Specific siRNAs were constructed as described previously (15). For transfection of 293T cells with siRNA, cationic lipid complexes were prepared by incubating 50 pmol of indicated siRNA with 5 l of DMRIE-C reagent (Invitrogen) in 200 l of Dulbecco's modified Eagle's medium for 20 min and were then added to the wells in a final volume of 0.75 ml with serum-free Dulbecco's modified Eagle's medium. 4 h later, 0.75 ml of Dulbecco's modified Eagle's medium containing 20% fetal calf serum was added to the cells.
The BRET Analysis for HIV-1 Protease Activity-The BRET assay used for the determination of HIV-1 protease activity is described in detail elsewhere (13). Briefly, 293T cells are transfected with hGFP2-p2/p7-hRLuc (1.5 mg), hGagPol (0.5 mg), and increasing concentrations of V5.LysRS. At 40 h after transfection, cells were harvested and analyzed for protein expression (see below) or 100,000 cells were plated in a microwell and were submitted to BRET analysis as follows. 5 mM DeepBlue C coelenterazine (PerkinElmer Life Sciences) was added to cells, causing the oxidation of the donor hRLuc and resulting in the emission of light of wavelength 395 nm. Part of the energy can be transferred non-radiatively to a codon-optimized and -humanized GFP2 (hGFP2) if the hRluc and the hGFP2 are in close proximity (Forster distance being about 10 -100 Å). The hGFP2 will then emit light at 510 nm. Both emissions are quantitated by sequential top-reading, using a combined luminometer and fluorescence plate reader (Fusion ϰ-FP apparatus, PerkinElmer Life Sciences). The BRET ratio is determined from the following equation: ((emission at 510 nm/emission at 395 nm) in cells expressing the hRluc and hGFP2 fusion proteins)) Ϫ ((emission at 510 nm/emission at 410 in cells expressing hRluc alone)). The following filters are used: the hRLuc emission, 410 nm bandpass of 80 nm, and the hGFP2 emission, 515 nm bandpass of 30 nm. In contrast to the first generation BRET analysis (16), the BRET analysis used here provides better resolution between the emission spectra of hRLuc and hGFP2, allowing for a straightforward ratiometric calculation to assess molecular proximity between hGFP2 and hRLuc and HIV-1 protease activity (13,17,18,21). The BRET ratio was found to be stable over several readings (evaluated within 5-8 min (13)). In some experiments, the expression levels of total hGFP2 and hRLuc were determined by direct measurements of total hGFP2 and luminescence levels on aliquots of transfected cell samples as follows. The hGFP2 total fluorescence was measured using Fusion ϰ-FP apparatus with an excitation filter of 400 nm and an emission filter of 510 nm. After the fluorescence measurement, the same cells were incubated for 10 min with coelenterazine H (Molecular Probes) at a final concentration of 5 M, and the total luminescence of cells was measured using the same instrument set up for bioluminescence readings. In contrast to DeepBlue C coelenterazine, coelenterazine H does not lead to energy transfer to hGFP2 and thus allows us to assess to hRluc expression without hGFP2 emission quenching. When expressed as a ratio, the total hGFP2:hRluc ratio was found to be similar in each experiment, indicating stable levels of expression of each protein.
Protein Analysis-Cellular and viral proteins were extracted with RIPA buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 2 mg/ml aprotinin, 2 mg/ml leupeptin, 1 mg/ml pepstatin A, 100 mg/ml phenylmethylsulfonyl fluoride). The cell and viral lysates were analyzed by SDS-PAGE (10% acrylamide) followed by blotting onto nitrocellulose membranes (Amersham Biosciences). Western blots were probed with monoclonal antibodies that are specifically reactive with HIV-1 capsid (Zepto Metrocs Inc.) and ␤-actin (Sigma), reverse transcriptase (anti-RTp66/p51, National Institutes of Health AIDS Research and Reference Reagent Program), and rabbit antibody to human LysRS (19). Detection of HIV proteins was performed by enhanced chemiluminescence (PerkinElmer Life Sciences) using as secondary antibody sheep anti-mouse for anti-RT, anticapsid, and anti-␤-actin and donkey anti-rabbit for anti-LysRS (Amersham Biosciences). Bands in Western blots were quantitated using the UN-SCAN-IT gel TM automated digitizing system (Silk Scientific). After BRET analysis, Western blots of cell lysates were probed with mouse monoclonal anti-V5 (Sigma) to detect LysRS C-terminally tagged with V5 and with antibody to glyceraldehydes-3-phosphate dehydrogenase (GAPDH), obtained from Research Diagnostics.

Overexpression of tRNA Lys in the Cell Increases the Viral Concentration of Both LysRS and RT-293T
cells were transfected with a plasmid containing HIV-1 proviral DNA alone (BH10) or a plasmid containing HIV-1 proviral DNA and a gene for either tRNA 3 Lys (BH10Lys3) or tRNA 2 Lys (BH10Lys2). Previous studies have shown that overexpression of tRNA 3 Lys results in an increase in its packaging into the virion, with a corresponding decrease in the viral concentration of the other tRNA Lys isoacceptors, tRNA 1 Lys and tRNA 2 Lys , i.e. the total viral tRNA Lys /virion remains constant (11,12). Viruses were purified, and Western blots of viral protein were probed with anti-RT, stripped, and reprobed with anti-CA (Fig. 1A). Quantitative analysis using the UN-SCAN-IT gel TM automated digitizing system indicated that there was ϳ1.7 times more RT/CA in virions produced from BH10Lys3 or BH10Lys2 than in wild-type virus.
This increase of RT in virions as a result of overexpression of tRNA 3 Lys or tRNA 2 Lys is not associated with an increase in the overall viral concentration of tRNA Lys (11,12). This suggests that some other molecule may be responsible for the increased viral RT. Since LysRS is also selectively packaged into HIV-1 during assembly (6) We have reported that transfection of 293T cells with small interfering RNA specific for LysRS (siRNA LysRS ) will reduce the incorporation of LysRS into virions 80%, and this reduction in viral LysRS is correlated with a similar reduction in the synthesis of new LysRS in the cytoplasm (15). Fig. 2B shows Western blots of lysates of viruses produced from 293T cells first transfected with either siRNA Luc (specific for luciferase) or siRNA LysRS and transfected 24 h later with the BH10 plasmid. Viruses were harvested 48 h after transfection of the BH10 plasmid, and the Western blots of viral protein were probed consecutively with anti-RT and anti-CA. The results, analyzed by UN-SCAN-IT gel TM automated digitizing system, indicate that the reduction in the cellular expression and viral incorporation of LysRS also results in an ϳ50% decrease in the viral concentration of RT.

Proteolysis of Viral Protein in the Cytoplasm Is Correlated with the Cytoplasmic Concentration of LysRS-Since
LysRS and GagPol are found in the same tRNA Lys packaging complex, the increased concentration of viral RT due to the increased incorporation of LysRS could reflect an increased recruitment of GagPol into this complex by the excess LysRS. However, this is not likely since we have previously shown that in a proteasenegative virion, overexpression of LysRS results in close to a 2-fold increase in both LysRS and tRNA Lys , without changing the GagPol:Gag ratio (11). An alternative explanation is that LysRS inhibits premature activation of viral protease in this cytoplasmic complex. The data in Figs. 3 and 4 support this premise. Fig. 3 shows Western blots of lysates of 293T cells that have been cotransfected with a plasmid coding for either proteasepositive HIV-1 DNA (A and C, BH10) or protease-negative HIV-1 DNA (B and D, BH10PϪ) and a second plasmid or small interfering RNA that will alter the expression of cytoplasmic LysRS. Western blots were probed with anti-CA, anti-RT, anti-LysRS, or anti-␤-actin. In A and B, the second plasmid is either the vector alone (pcDNA3.1) or this vector containing the gene for LysRS (pcDNALysRS). The results in A, which are quantitated in E, show that there is less proteolysis of viral proteins upon overexpression of LysRS in the cytoplasm, i.e. the cytoplasmic Gag/CA and GagPol/RT ratios are ϳ4.5and 8-fold greater, respectively, when the LysRS/actin ratio is increased. B is a control showing the cytoplasmic viral protein pattern when no viral protease is present.
The cells represented in C and D have been transfected with either siRNA Luc or siRNA LysRS as well as either BH10 (C) or BH10PϪ (D). The results in C, which are quantitated in E, show that there is more proteolysis of viral proteins when the concentration of cytoplasmic LysRS is decreased, i.e. when the LysRS/␤-actin ratio is reduced ϳ90%, the cytoplasmic Gag/CA and GagPol/RT ratios are reduced ϳ80 -85%. D is again a control showing the cytoplasmic viral protein pattern when no viral protease is present, indicating that the proteolysis of the viral proteins is not due to the activation of cellular proteases due to the reduced LysRS in the cell.

Effect of LysRS Expression on Viral Protease Activity Using
BRET Analysis-We next examined the effect of LysRS concentration upon HIV-1 protease activity using BRET to assay for HIV-1 protease activity in living cells (13). BRET analysis has been used to study Gag binding partners in HIV-1, homoand heterodimerization of integral membrane receptors, protein-protein interactions during transcription, and more recently, in an ubiquitination biosensor assay (16 -18, 21). The assay takes advantage of the physical distance between a donor and acceptor molecule, and when these are within close proximity (between 10 -100 Å), a resonance energy transfer (RET) occurs between the donor and acceptor pair. To initiate this reaction, a membrane soluble coelenterazine, DeepBlueC (PerkinElmer Life Sciences), is added to cells in culture. In the presence of oxygen, humanized sea pansy Renilla reniformis luciferase (hRLuc) catalyzes the DeepBlueC into coelenteramide with concomitant light emission (l 1 em ϭ 395 nm). The acceptor of this emission is a codon-optimized (humanized) green fluorescent protein 2 (hGFP2) that is engineered to maximally absorb l 1 em. Excitation of hGFP2 by RET results in an emission of green light (l 2 emϭ510nm). The broad spectral emission spectra exhibited by these molecules enable a 1 is a vector plasmid, whereas pcDNALysRS is this plasmid containing the cytoplasmic form of human LysRS cDNA. This cDNA is tagged at the C terminus with the V5 epitope and has a slower electrophoretic mobility than endogenous LysRS (20). 48 h post transfection with BH10, cells were lysed in RIPA buffer, and proteins in the cell lysates were resolved by Western blotting and probed with anti-CA, anti-RT, anti-LysRS, and anti-␤-actin. Detection of proteins was performed by enhanced chemiluminescence (PerkinElmer Life Sciences) using as secondary antibodies anti-mouse (for anti-capsid, anti-reverse transcriptase, and anti-␤-actin) and donkey anti-rabbit (for anti-LysRS), obtained from Amersham Biosciences. A and B, cells were cotransfected with either BH10 or BH10P-and with either pcDNA3.1 or pcDNALysRS. C and D, cells were cotransfected with either BH10 or BH10P-and with either siRNA Luc or siRNA LysRS . E, quantitation of bands was performed using UN-SCAN-IT gel TM automated digitizing system, and ratios are listed in E. straightforward ratiometric calculation (16 -18, 21).
To determine whether LysRS expression affects viral protease activity in live cells, the amino acids that encode the p2/p7 protease cleavage site in Pr55 Gag were fused in-frame with the donor protein, hRLuc, and the acceptor protein, hGFP2, to create hGFP2-p2/p7-hRLuc (Fig. 4A). 293T cells were cotransfected with this construct, a plasmid coding for codon-optimized protease-positive Gag and GagPol (hGagPol (22)), and increasing amounts of pcDNALysRS. 40 h after transfection, cells were exposed to the membrane-diffusible substrate for luciferase, DeepBlueC, and analyzed by BRET. After BRET analysis, cells were lysed, and Western blots of cell lysates were probed with anti-V5 and anti-GAPDH to show the increased ratio of LysRS/GAPDH (Fig. 4B).
BRET analysis is shown in Fig. 4C. A close association between hGFP2 and hRLuc will result in a maximum BRET ratio (lane 6). However, if hGFP2 and hRLuc are separated as a result of cleavage by the viral protease, there will be a decreased BRET ratio (lane 2). Fig. 4C shows that the BRET ratio increases with increasing cellular concentrations of Ly-sRS (lanes 2-5), indicating the inhibition of hGFP2/hRLuc cleavage. Fig. 4C, lanes 6 -8, represent control experiments in which cells are either cotransfected with the plasmid coding for hGFP2-p2/p7-hRLuc and a plasmid (hGagPol⌬FS⌬PR (22)) coding for protease-negative codon-optimized GagPol (lane 6), hRLuc alone (lane 7), or hGFP2 alone (lane 8). The BRET ratio is maximal when no cleavage occurs (lane 6), and in this experiment, the ratio is reduced by 60% when only endogenous levels of LysRS are expressed (lane 2). Thus, these BRET data also indicate that LysRS has an inhibitory effect upon viral protease activity. DISCUSSION GagPol incorporated into the assembling Gag particle through its interaction with Gag (3,4,23,24). The concentration of GagPol at the cell membrane is believed to facilitate the concentration of GagPol molecules, allowing for GagPol dimerization, a prerequisite to protease activation. Premature dimerization of GagPol at the wrong cellular site could result in the production of processed viral proteins that will not take part in viral assembly. GagPol interacts with multimeric Gag (5), and because pre-plasma membrane complexes of Gag/GagPol may exist in the cytoplasm, means may be necessary for preventing premature protease dimerization and activation in such complexes until they reach the proper cellular compartment.
Our data indicated that the interaction between Gag and GagPol is not sufficient to prevent premature protease activation and that another member of the assembly complex, LysRS, may also help stabilize GagPol protease during the transition to the plasma membrane. In the virion, estimates for Gag are ϳ1500 -1800 molecules/virion (1), and based on estimated differences in translation rates of Gag and GagPol, there may be ϳ150 -200 molecules of GagPol. This is considerably more than the estimated of 20 -25 molecules LysRS/virion (25), and unsaturated LysRS binding sites may exist in the Gag/GagPol/ RNA complex. The overexpression of LysRS results in an approximate 2-fold increase in LysRS incorporation into HIV-1 (20) without any increase in GagPol incorporation (11), and the limited increase in LysRS packaging may reflect either limited cellular LysRS overexpression or some other limiting factor. These results suggest that within the experimental conditions used here (such as overproduction of virions), there may exist in the cell multiple assembly complexes of varying stabilities due to limiting amounts of cellular factors such as LysRS.
The mechanism by which LysRS blocks proteolytic activity is not known. It could be inhibiting the viral protease activity generally, or it could act to block access to cleavage sites by the protease. The first cleavage is believed to occur at the p2/NCp7 site (26), the site examined by BRET. Although it is clear that proteolytic activity at later cleavage sites is also inhibited (such as the ones producing mature CAp24 and RTp66/p51), later cleavages might be prevented as a result of preventing the first cleavage. Nevertheless, it seems unlikely that the relatively small number of LysRS molecules associated with virions could directly protect the much larger number of cleavage sites or protease molecules. We have been unable to detect an interaction between Pol and LysRS using coimmunoprecipitation studies (data not shown), and we suggest that the most likely cause of inhibition of protease activity may be changes induced by LysRS in the GagPol conformation, which might result in either inhibiting protease activity or blocking one or more cleavage sites in GagPol.
The changes observed in the RT incorporation into virions induced by alterations in the cellular concentration of LysRS (Fig. 2) are consistent but not large. Increases and decreases in cellular LysRS cause increases and decreases in the RT/CAp24 ratios of 1.77 and 0.56, respectively. On the other hand, the data in Fig. 3 show that the cellular concentration of LysRS has a much larger influence on the amount of Gag or GagPol processed in the cytoplasm. Thus, a 5-fold increase in the cytoplasmic concentration of LysRS produces a 4.6-and 7.8-fold increase in the Gag/CAp24 and GagPol/p66 ratios, respectively. Similarly, an almost 10-fold decrease in cytoplasmic LysRS shows a 4-and 5-fold decrease in Gag/CAp24 and GagPol/p66 ratios, respectively. Thus, an increase in the amount of premature processing of GagPol may have less effect upon the amount of RT incorporated into virions (i.e. RT/CAp24) because there is also less Gag produced.
Thus, the data obtained from the Western blots shown in Figs. 1-3 indicate inhibition of viral protease activity by LysRS, and as shown in Fig. 4, this conclusion is supported using a very different experimental technique, BRET. In this technique, HIV-1 protease activity is detected by its ability to act upon the cleavage site between Gag p2 and p7 that is present in the hGFP2-p2/p7-hRLuc molecule, thus separating donor hRLuc from acceptor hGFP2. Since there is no evidence that this molecule can be packaged into virions, it is implied that it is the cleaved viral protease, free of the Gag/GagPol complex, that is acting upon hGFP2-p2/p7-hRLuc.
Other work has shown that the infectivity of the HIV-1 population increases 2.5-3-fold when LysRS and tRNA Lys are overexpressed in the cell. The viral population increases its incorporation of LysRS and tRNA Lys , and there is also an increase in tRNA 3 Lys annealing to viral RNA (11). Although this increase in tRNA 3 Lys annealing had been thought to be the prime factor responsible for the increased infectivity, it is now clear that other factors, such as the decreased premature proteolysis of Gag and GagPol studied here, may also contribute to increased infectivity of the viral population.